This invention relates to the incorporation of a wide variety of therapeutically useful substances into mammalian red blood cells (RBC), which could not previously be accomplished without unacceptable losses of RBC contents and/or integrity. More particularly, the method of the present invention makes possible the introduction or incorporation of agents into RBC, such as peptides, purine and purine analogs, pyrimidine and analogs thereof, chemotherapeutic agents, antibiotic agents, and unnatural analogs of the nucleic acid bases adenine, guanine, cytosine and thymine. These and other water soluble substances, may be used for a desired slow continuous delivery or targeted delivery when the treated RBC carrier is later injected in vivo.
The method of this invention has particular utility when used in the so-called "osmotic pulse" mechanism taught in U.S. Pat. No. 4,478,824, issued Oct. 23, 1984, to the present applicants, the disclosure of which is incorporated by reference herein. The method of this patent involves incubating a packed RBC fraction in a solution containing a compound (such as dimethyl sulfoxide or glycerol) which readily diffuses into and out of cells, rapidly creating a trans-membrane osmotic gradient by diluting the suspension of RBC in the solution with a near-isotonic aqueous medium containing an anionic agent to be introduced (such as a phosphorylated inositol) which may be an allosteric effector of hemoglobin, thereby causing diffusion of water into the cells with consequent swelling thereof and increase in permeability of the outer membranes of the cells, and maintaining the increase in permeability for a period of time sufficient only to permit transport of the anionic agent into the cells and diffusion of the compound out of the cells.
However, the method of this patent is of limited effectiveness where the desired agent to be incorporated within the cells is not anionic or is anionic or polyanionic but is not present in the near-isotonic aqueous medium in sufficient concentration to cause the needed increase in cell permeability without cell destruction. The present invention constitutes a discovery that non-anionic agents can be introduced into RBC by addition to the near-isotonic aqueous medium of an effective amount of a water soluble polyanion, as hereinafter defined, which acts as a "co-factor" in causing the non-anionic agent or agents in the aqueous medium to be incorporated in the cells with optimum efficiency without unacceptable loss of cell contents. In effect, the polyanion co-factor provides a "free ride" for non-anionic agents through the outer membranes of the cells and into the cells during the relatively brief time period of increased permeability. This co-factor concept is also applicable for incorporation of anionic reagents whose introduction into RBC is desired in low concentrations not sufficient to provide the essential increase in permeability and transport into intact cells as described above. The term "effectively non-anionic" is thus used hereinafter to designate a desired agent to be introduced which is non-anionic, or which is anionic or polyanionic but is present in such low concentration in the near-isotonic aqueous medium as to be incapable of acting as a co-factor (i.e. less than the functional equivalent of 1 millimolar of inositol hexaphosphate, as explained below).
In a further embodiment, for use where the desired agent to be introduced would react with a polyanion co-factor to produce an insoluble product (or where use of a polyanion is otherwise undesirable), the present invention provides the application of shear induced resealing of RBC within a short period of time after the dilution step, in place of addition of a polyanion.
R. S. Franco et al. have disclosed part of the subject matter of the above-identified Pat. No. 4,478,824 in an article in Life Science, 32: 2763-2768 (1983).
R. E. Benesch et al., in Biochemistry, 16: 2594-1597 (1977) have shown that inositol hexaphosphate (IHP) binds to hemoglobin at the 2, 3 diphosphoglycerate site and causes a decrease in oxygen affinity. In addition to the osmotic pulse method for incorporating IHP into red cells disclosed in the above patent, a liposomal method is disclosed by Gerosonde in Blut, 39: 1-7 (1979), and a lysis-resealing method is disclosed by B. Teisseire et al. in Adv. Exp. Med., 180: 673-677 (1984).
P. C. Anderson et al., Biophys. J., 20: 181-191 (1977) describe two-phase release of hemoglobin for a hypotonic lysis. In the first phase the time to initial lysis is a function of the osmotic driving force and for hypotonic lysis is limited by the osmolarity of the cell interior.
M. P. Sheetz, in Semin. Hematol., 20: 175-188 (1983) showed that IHP at millimolar concentrations dissociates isolated red cell cytoskeletons and appears to act primarily on the spectrin-actin-band 4.1 complex. An earlier article by M. P. Sheetz et al., J. Biol. Chem., 255: 9955-9960 (1980) disclosed that exposure of intact membranes to a high concentration of polyphosphate did not result in elution of spectrin, from which it was inferred that this form of skeletal disruption does not interfere with binding to the bilayer membrane.
P. Heubsch et al., J. Cell. Physiol., 122: 266-272 (1985) showed that cytoskeletal detachment occurred in osmotically swollen cells and that with extreme changes in size the bilayer membrane is released from a deformational constraint which is present under normal conditions.
U.S. Pat. No. 4,224,313, issued Sept. 23, 1980, to U. Zimmermann et al. discloses a process for preparing a mass of loaded cells suspended in a solution by increasing the permeability of the cell membranes by osmotic pressure or an electric field, or both, incorporating loading material by passage from a solution through the membranes of increased permeability, restoring the original permeability by healing the membranes by regeneration effect, and separating the cells from the solution in which they were suspended. The loading material in solution includes a pharmaceutical substance having a capability of reacting chemically or physically with substances in solution outside the cell and which when incorporated in the cell would prematurely destroy the cell membranes, and at least one blood-compatible sugar and protein capable of providing hydrogen-bridge-bonding with the pharmaceutical substance or of entering into covalent bonds therewith, thereby inhibiting the reaction of the pharmaceutical substance with the cell membranes.
A description of experimental work leading to the present invention is contained in an article by R. S. Franco et al., J. Cell. Physiol., 129: 221-229 (1986).
Other publications by applicants include R. S. Franco et al., Am. J. Hematol., 17: 393-400 (1984) and R. S. Franco et al., Blood, 66 [suppl]: 277a (1984).
Other publications of which applicants are aware include U.S. Pat. No. 4,321,259, issued Mar. 23, 1982, to Y.-C. Nicolau et al.; Parsons et al. Chem. Abst., 91: 68355d (1979); Zimmermann et al. Chem. Abst., 94: 90235y (1981); Zimmermann et al. chem Abst., 89: 1355851m (1978); and Zimmermann et al. Chem. Abst., 86: 34228w (1977).
Despite the extensive work in the field of the present invention, as evidenced by the publications acknowledged above, there is still no method known in the prior art which provides introduction of non-anionic agents into RBC without unacceptable losses of RBC contents and without use of toxic or otherwise undesirable reagents and other materials in the process.
It is a primary objective of the present invention to provide a method which fulfills the above need.